None of the references described or referred to herein are admitted to be prior art.
Various industrial and commercial processes require the accurate measurement of optical electromagnetic emissions of differing wavelengths.
For example, in the field of nucleic acid diagnostics, to detect different nucleic acids of interest, different probes configured to hybridize to different nucleic acids, each of which may provide detectibly different fluorescent emission signals, can be used. Different probes configured to hybridize to different targets can be formulated with fluorophores that fluoresce at a predetermined wavelength when exposed to excitation light of a prescribed excitation wavelength. Assays for detecting different target nucleic acids can be performed by alternately exposing sample material to different excitation wavelengths and detecting the level of fluorescence at the wavelength of interest corresponding to the probe for each target nucleic acid of interest. Parallel processing can be performed using different signal-detecting devices constructed and arranged to periodically measure signal emissions during the assay process, and with different signal-detecting devices being configured to generate excitation signals of different wavelengths and to measure emission signals of different wavelengths to thereby detect the different nucleic acid of interest. Because the probe hybridizes to the targeted sequence or its complement in a manner permitting detection of a signal indicating the presence of the targeted sequence in a sample, the strength of the fluorescent signal is proportional to the amount of target sequence or its complement that is present in the sample.
In general, an optical measurement device (“OMD”) configured to measure an optical emission signal (e.g., detect the presence or absence of and/or determine the intensity of) will include components for generating an excitation signal, directing the excitation signal at a target, receiving an optical emission signal from the target, and generating an electrical signal, such as a current and/or voltage, corresponding to the strength or intensity of the emission signal received. Such an OMD may comprise, for example, a fluorometer configured to direct an excitation signal of a prescribed wavelength at a target and generate an output signal, such as a current or voltage, based on receipt of a fluorescent emission signal of a prescribed wavelength from the target. Such an OMD may comprise a light-emitting element, such as a light-emitting diode (LED), a light-detecting element, such as a photodiode, optic elements, such as one or more lens(es), filter(s), mirrors, optical collimators, optical wave guides (such as optic fibers), beam splitters, etc., and integrated circuits. The OMD may include a housing or other structure on which components of the OMD are supported. Such a housing may provide a window through which excitation light passes out of the housing and emission light passes into the housing, but the housing may otherwise provide a light-tight environment to minimize the influence of stray light on the emission signal detection. The optic elements may define optic paths from the light-emitting element to the window and from the window to the light-detecting element.
Suitable signal-detecting devices include fluorometers, such as a fluorometer described below. An automated nucleic acid diagnostic instrument may be configured to process numerous samples carried in multiple receptacles, and each fluorometer may be configured to take fluorometric readings from the receptacles as they are indexed past the fluorometer, or as the fluorometer is indexed past the receptacles, for example, once every 3 seconds. Thus, 1200 times for each hour of operation of the instrument, each fluorometer generates an excitation signal that is directed at the sample receptacle and measures the emission signal emitted by the contents of the receptacle, generating an electrical signal that is proportional to the intensity of the emission signal.
OMDs, such as fluorometers, are susceptible to generating false, poor, and/or inconsistent readings for a number of reasons, including inherent differences between individual fluorometers due to the manufacturing process, malfunctioning of the OMD, and accumulation of debris in the system (primarily on or around the optic element). An OMD assembly may include numerous components and tolerances in the construction and installation of such components may exist from one OMD to the next. For example, system to system variability may be created by the stacked tolerances relating the construction and installation of light sources, optic fibers, lenses, filters, mirrors, etc. Such structural variability can lead to signal variability. Thus, the signals of the OMDs can be calibrated, i.e., standardized or normalized, to the signals of a “standard” OMD detecting an emission signal from a known emission source.
A malfunction (device failure and/or deteriorated performance) by an OMD during operation of the instrument or miscalibration of the OMD will cause errors in the optical readings generated by that OMD and thereby cause errors in the diagnostic results. Such malfunctions may be due to mechanical and/or electrical failures that occur during operation of the OMD. While operation of the OMDs can be checked during routine maintenance of the instrument, such opportunities for testing are rare, since the testing can only be performed when the instrument is shut down. In some instances, the instrument is operated continuously for extended periods of time for maximum throughput. Therefore, it becomes impractical and non-cost-effective to repeatedly shut the instrument down to perform OMD functionality testing.
Calibrating an OMD, such as a fluorometer, and/or monitoring the performance of the OMD involves typically generating an emission signal (a fluorescent reference emission) of known intensity and/or wavelength. The reference emission is detected by the OMD to be calibrated or monitored and the signal generated by the OMD from the reference signal is compared to the signal to be expected from the reference emission. For calibration, if the actual and expected signals do not agree, the OMD may be adjusted as necessary, e.g., by adjusting electronic gains in the signal processing electronics, so that the signal generated by the OMD matches the expected signal.
In the past, different mechanisms have been employed for generating reference emission signals for calibrating and/or monitoring fluorometers and other OMDs.
For example, a reference emission could be generated by a light source providing an optical signal of known intensity as well as, optionally, providing a referencing signal of a known wavelength. Such alight source may comprise a light emitting diode, a laser, or a white light and appropriate filters. Such devices are difficult and expensive to build and maintain. In addition, the output of a light source may not be stable, so that a reference emission generated b the source may not be stable. Furthermore, such devices may be relatively large and bulky and may not be suitable for calibrating or testing the OMD in its normal operating environment thereby requiring that the OMD be removed from an instrument or system in which it is employed so that it can be tested and re-calibrated.
Another mechanism for generating a reference emission is the use of controlled sources that generate known optical emission signals. Such sources may comprise fluorescent sources, such as liquid dyes. Such dyes can be placed into a receptacle, e.g., a multi-well plate, and placed into a diagnostic instrument for detection by the OMD and the signal generated by the OMD can be compared to an expected signal from the fluorescent source. Such fluorescent sources can, however, be unstable and often have special storage requirements and pre-use preparation procedures. For example, liquid dyes may need to be stored in a frozen state and require special preparation procedures prior to their use. In addition, such sources may be unstable and may need to be used within a relatively short period of time following their preparation. Fluorescent dyes may also be susceptible to photo-bleaching, whereby repeated exposure of the fluorescent source to an excitation light signal may alter the emission signal over a period of time.
A third mechanism for generating a reference emission is to use emissive plastics, such as fluorescent plastics. Typical plastics used to date fluoresce at certain specific wavelengths (i.e. colors), and thus different plastics or differently-colored plastics are required for testing different fluorometers configured to detect emissions of different wavelengths. In addition, fluorescent plastics used today can be unstable and degrade over time and are susceptible to photo-bleaching. Thus, the reference emission signals generated by such plastics can be degraded over time and/or after repeated exposures to an excitation signal.
Accordingly, a need exists for means and methodologies for periodically confirming the proper functionality of the OMDs during the operation of the instrument as well as for calibrating or standardizing multiple OMDs so that they generate consistent readings.